Lipofectamine-Mediated CRISPR-Cas9 Delivery Targeting the mecA Gene in Methicillin-Resistant Staphylococcus aureus: A Novel Approach to Reverse Antibiotic Resistance

Abstract

Background: Healthcare-associated (HA) infections caused by methicillin-resistant Staphylococcus aureus (MRSA) continue to be a big problem worldwide. The mecA gene is responsible for the production of PBP2a which is responsible for the resistance to all beta-lactam antibiotics. There are currently very little alternative therapeutic options as the treatment is becoming limited. The advantage of CRISPR-Cas9 technology lies in the ability to target genes with precision, however, there are technical delivery challenges in the application of this technology in bacterial pathogens. Objective: The aim of this study was to assess the efficacy of CRISPR-Cas9 delivery by liposomes with Lipofectamine and its ability to knock out the mecA gene in MRSA and make them susceptible to beta-lactam antibiotics. Methods: MO of MRSA ATCC 43300 was electroporated with a Cas9-sgRNA RNP complex using Lipofectamine CRISPRMAX with a mecA-targeting guide sgRNA designed using CHOPCHOP. An antibacterial activity was determined (CFU, growth curves, biofilm), gene targeting (PCR, Sanger sequencing), resistance reversal (MIC, disk diffusion) and safety (MTT, hemolysis). All experiments were repeated three times (n=3), data presented as mean ± SD and analyzed by One Way Analysis of Variance (ANOVA) with Tukey's post hoc test (p<0.05). Results: The treatment group showed reduction of CFU level, delay of growth and 60-70% inhibition of the development of biofilm which was significant (p=0.008). This was demonstrated by PCR with reduced intensity of mecA amplicon (1/3 of controls) and by Sanger sequencing which revealed the presence of indels in 72% of the clones. MIC dropped from 64 to 4 μg/mL (p<0.001), and inhibition zones increased from 0 to 22.3 ± 1.8 mm. Cell viability exceeded 80% (87.4 ± 3.2%), with hemolysis below 5% (3.1 ± 0.4%). Conclusion: Lipofectamine-mediated delivery of CRISPR-Cas9 effectively targets the mecA gene, reverses antibiotic resistance, and demonstrates an acceptable safety profile in vitro. These findings provide a proof-of-concept for this approach and warrant further investigation in appropriate in vivo models.